This application is a xc2xa7371 of PCT application No. PCT/AU99/00659, filed Aug. 13, 1999.
This invention relates to a method of isolating monoclonal antibody inhibitors and reagents derived therefrom and other inhibitors of cytokine binding including monoclonal antibodies and reagents derived therefrom and small molecules capable of inhibiting binding of GM-CSF, IL-3 and IL-5 to the common beta receptor subunit.
Human interleukin (IL)-5, IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are cytokines involved in hemopoiesis and inflammation (Metcalf; 1986). All three cytokines stimulate eosinophil production, function and survival (Metcalf; 1986) and therefore have the ability to influence inflammatory diseases such as asthma, atopic dermatitis and allergic rhinitis where the eosinophil plays a major effector role. IL-5, being the eosinophil specific cytokine, has received most of the initial attention with IL-5 mRNA and protein levels noted to be elevated in lung tissue and bronchoalveolar lavage (BAL) fluid from symptomatic asthma patients (Fukuda et al 1994). Correlation between IL-5 levels and allergen challenge and disease activity have also been seen (Sur et al, 1996). It is becoming apparent, however, that not only IL-5 but also GM-CSF and IL-3 play a role in eosinophil production and activation in asthma as there is evidence of both GM-CSF and IL-3 being synthesized at sites of allergic inflammation (Bagley et al, 1997b; Allen et al 1997). It is possible that expression of these cytokines contributes to the total number of infiltrating eosinophils and the degree of eosinophil activation. Alternatively, they may be responsible for different phases of eosinophil infiltration. Recent kinetic data from patients undergoing antigen challenge showed that IL-5 levels increased between days 2-7 post challenge, whilst GM-CSF peaked at day 2, and remained elevated throughout day 16. Furthermore, GM-CSF detection extended beyond the site of allergen challenge.
IL-5, GM-CSF and IL-3 stimulate eosinophils and other normal and cancer cells by binding to cell surface receptors that comprise a ligand-specific xcex1 chain and a xcex2 chain which is shared by the three receptors (xcex2c) (Bagley et al 1997a). Binding to each receptor xcex1 chain is the initial step in receptor activation, however, engagement of either xcex1 chain alone is not sufficient for activation to occur. Recruitment of xcex2c by each ligand: xcex1 chain complex follows, a step that has two major functional consequences: firstly, it allows the binding of IL-5, GM-CSF and IL-3 to become essentially irreversible; and secondly, it leads to full receptor activation (Bagley et al 1997a). Since xcex2c is the major signalling component of these receptors its engagement leads to the activation of JAK-2, STAT-5 and other signalling molecules culminating in the full plethora of cellular activities commonly associated with either IL-5, GM-CSF and IL-3 stimulation such as eosinophil adherence, priming for degranulation and cytotoxicity, and prolongation of viability (Bates et al, 1996).
In order to block or antagonize the activity of eosinophil-activating cytokines in vivo three major approaches are being tried. One of them utilizes antibodies to the implicated cytokines. For example, antibodies to IL-5 are being used in an animal model of allergen-induced asthma and have shown to have a relatively long lasting effect in preventing eosinophil influx into the airways and bronchial hyperresponsiveness (Mauser et al, 1995). A second approach relies on IL-5 or GM CSF mutants which can bind to the respective xcex1 chains with wild type affinity but which have lost or shown reduced ability to interact with human xcex2c. IL-5 mutants such as E13Q, E13K and E13R, and the human GM-CSF mutant E21R directly antagonize the functional activation of eosinophils by IL-5 or GM-CSF respectively (Tavernier et al 1995; McKinnon et al 1997; Hercus et al 1994b). However, at least in the case of E13K, eosinophil survival is not antagonized and in fact this mutant is able to support eosinophil survival (McKinnon et al 1997). A third approach involves the use of soluble receptor xcex1 chains which can sequester circulating cytokines. However, this carries the risk of a cytokine: receptor xcex1 chain complex potentially interacting with surface-expressed xcex2c and triggering receptor activation. The common theme amongst these approaches is that they tackle a single receptor system involving either IL-5, GM-CSF or IL-3 leaving the other two eosinophil-acting cytokines unaffected. Although the concomitant administration of IL-5 and GM-CSF antagonists may be considered, this may be clinically impracticable.
An alternative approach to blocking eosinophil-activating cytokines involves targeting the common xcex2 chain of their receptors. Although xcex2c does not directly bind IL-5, GM-CSF or IL-3 alone, it does bind to these cytokines complexed to the appropriate receptor xcex1 chain. Lopez et al in WO 97/28190, which is incorporated herein by reference in its entirety, have identified the major binding sites of, xcex2c for the IL-5:IL-5Rxcex1, GM-CSF:GM-CSFRxcex1 and IL-3:IL-3Rxcex1 complexes. Significantly, these sites are utilized by all three complexes and comprise the predicted Bxe2x80x2-Cxe2x80x2 loop and Fxe2x80x2-Gxe2x80x2 loop in xcex2c. Thus targeting xcex2c is not only desirable but also feasible, with the added potential to allow the simultaneous inhibition of IL-5, GM-CSF and IL-3 action by a single agent. These workers have shown that certain mutants in the Bxe2x80x2-Cxe2x80x2 and the Fxe2x80x2-Gxe2x80x2 loop fail to bind IL-5, GM-CSF and IL-3.
The present invention results from the isolation of a monoclonal antibody (BION-1) raised against the membrane proximal domain (domain 4) of xcex2c which is able to block the production and activation of human eosinophils stimulated by IL-5, GM-CSF or IL-3 and blocks the growth of leukaemic cell lines. This MoAb was able to block the high affinity binding of all three cytokines to eosinophils by binding to residues in the predicted Bxe2x80x2-Cxe2x80x2 and Fxe2x80x2-Gxe2x80x2 loops of xcex2c, and prevented receptor dimerization and xcex2c phosphorylation. It was found that raising an antibody capable of blocking the binding of all three cytokines was possible by screening monoclonal antibody-expressing hybridoma cell lines arising from immunising mice with cells expressing only domain 4 of xcex2c and lacking domains 1 to 3 and expressing domain 4 and the transmembrane and cytoplasmic regions.
Additionally this finding is likely to have implications for other members of the cytokine receptor superfamily some of which are shared subunits in a given subfamily (that is they bind several cytokines), and some which are ligand specific and bind to only one cytokine. The receptor a-chains for GM-CSF, IL-3 and IL-5 and xcex2c belong to the rapidly expanding cytokine receptor superfamily. Within this superfamily several sub-families are now emerging that are characterized by the sharing of a communal receptor subunit by multiple ligands: gp130 acts as an affinity converter and signal transducer for IL-6 (Hibi et al., 1990; Taga et al., 1992), IL-11 (Hilton et al., 1994), oncostatin M (Liu et al., 1992), ciliary neurotrophic factor, leukaemia inhibitory factor (LIF) (Ip et al., 1992) and cardiotrophin-1(Pennica et al., 1995); the LIF receptor (LIFR) also binds ciliary neurotrophic factor (Davis et al., 1993), cardiotrophin-1 (Pennica et al., 1995) and oncostatin M in addition to LIF (Gearing et al., 1994); IL-2R xcex2 supports affinity conversion and signalling of IL-2 and IL-15 (Giri et al., 1994); IL-2R xcex3 chain affinity converts IL-2 (Takeshita et al., 1992), IL-4 (Russell et al., 1993), IL-7 (Noguchi et al., 1993), IL-9 (Kimura et al., 1995) and IL-15 (Giri et al., 1994); evidence also suggests that IL-4 and IL-13 share a receptor component (Zurawski et al., 1993) and this subunit has recently been cloned (Hilton et al., 1996). It is not known which residues in gp130, LIFR and IL-2R xcex2 and xcex3 chains are important for ligand binding or indeed whether different ligands share or have unique sets of binding determinants on these communal receptor subunits. Because these common subunits are vital for transducing signals by several ligands, the possibility arises that interfering with the ability of these common subunits to bind ligand or to form homodimers may affect the action of more than one ligand.
Clear similarities in structure between xcex2c and other cytokine receptors have been recognised and similarities in at least part of the binding site, the Fxe2x80x2-Gxe2x80x2 loop, have been identified in Lopez et al in WO 97/28190. Accordingly it is an expectation that the means employed by the inventors to obtain a monoclonal antibody that inhibits binding of IL-3, GM-CSF and IL-5 will also lead to the isolation of monoclonal antibodies that inhibit binding of other cytokines to their respective receptors.
In a broad form of a first aspect the invention could be said to reside in a method of isolating a monoclonal antibody capable of inhibiting any one of IL-3, GM-CSF and IL-5 binding to the common receptor xcex2c, or a monoclonal antibody capable of inhibiting a cytokines binding to a receptor analogous to xcex2c, said method comprising the step of immunising an animal with a cytokine receptor or portion of a cytokine receptor containing the critical binding site which portion might include the extracellular domain 4 or analogous domain in the analogous common receptor or part thereof, isolating antibody producing cells from said animal and fusing antibody producing cells with a myeloma cell line, screening for a cell line that produces an antibody of the desired type.
The immunisation may involve introducing a cDNA clone of a portion of or all of the common receptor including the extracellular domain 4 or analogous domain in the analogous common receptor or part thereof, into a cell and proliferating said cells to form a recombinant cell line, inoculating an animal with said recombinant cell line, isolating antibody producing cells from said animal and fusing the antibody producing cell line with a myeloma cell line to form a hybridoma cell line, screening for a hybridoma cell line that produces an antibody that binds to the recombinant cell line but not to the parent, and then testing for inhibition against all three cytokines. In one form the cell into which the cDNA clone is introduced is mammalian and one commonly used mammalian cell line is a COS cell.
The cDNA may encode a full or partial portion of domain 4 when it is in a configuration where the Fxe2x80x2-Gxe2x80x2 loop and/or the Bxe2x80x2-Cxe2x80x2 loop is in its native shape. The data below show that cDNA encoding substantially only domain 4 of the extracellular portion of xcex2c as well as the transmembrane and the intracellular portions maintains these sites in a sufficiently integral conformation so that an antibody raised thereagainst will give the inhibition sought. It is postulated that the same will be the case for analogous receptors for the cytokine superfamily. This method should be distinguished from immunising with the whole receptor since the extracellular domain 4 is likely to be covered or masked by other domains in the whole receptor.
xcex2c has two repeats of the cytokine receptor module (CRM), each of which has two discrete folding domains (CRDs), so that in total xcex2c has 4 domains hence named domains 1 to 4 (xcex21 to 4). It is postulated that domain 2 of any CRM may be an equivalent of domain 4 and therefore domain 2 may be used in the immunisation.
In addition the domain 4 of xcex2c or equivalent domain in other cytokine receptors may be expressed in isolation in a microbial host such as Escherichia coli and used to immunise animals for developing monoclonal antibodies.
The analogous receptor may be any one of the cytokine superfamily receptors but not limited to the group comprising xcex2c, LIFR, gp130, IL-2Rxcex2, IL-4R/IL-13R, IL-2Rxcex3, IL-3Rxcex1, EPOR, TPOR and OBR.
It will be understood that in one specific form of this aspect of the invention the method is used to isolate a monoclonal antibody that inhibits cytokine binding to a common receptor subunit. The common receptor is envisaged to be selected from the group of receptors acting for more than one cytokine including but not limited to gp130, LIFR, IL2Rxcex2/IL2Rxcex1, and IL-4R/IL-13R in addition to xcex2c.
It will also be understood that the invention encompasses monoclonal antibodies or fragments thereof produced as a result of this first form of the invention.
In a broad form of a second aspect the invention could be said to reside in a monoclonal antibody, or fragments thereof capable of inhibiting the binding of the three cytokines IL-3, GM-CSF and IL-5 to the xcex2c receptor.
The degree of inhibition may range from complete inhibition to moderate inhibition, which inhibition will of course be dependent on the amount of monoclonal antibody or fragments thereof added to inhibit and the relative affinity of the antibody or fragment thereof to the xcex2c.
The extent of inhibition of respective ones of the three cytokines is not necessarily identical and may vary, so the different cytokines may be inhibited from binding to different degrees.
The antibody fragments may be larger portions such as Fab fragments or much smaller fragments of the variable region. These fragments may be used as separate molecules or alternatively may form part of a recombinant molecule which is then used for therapeutic purposes. Thus for example the monoclonal antibody may be xe2x80x9chumanisedxe2x80x9d by recombining nucleic acid encoding the variable region of the monoclonal antibody with nucleic acid encoding non-variable regions of human origin in an appropriate expression vector.
The inhibition preferably leads to blocking of at least one function of all three cytokines. One of the benefits that is proposed to be derived from these antibodies or antibody fragments is their use in modifying cells stimulated by one of the three cytokines, and more in one specific form modifying the activity of the three cytokines is proposed to impact greatly on eosinophil function. Therefore preferably the activity leads to inhibition of stimulation of effector cell activation and where the antibody or fragment thereof is to be used for treatment of asthma leads most preferably to inhibition of IL-5, IL-3 and GM-CSF mediated eosinophil activation. It will be understood however that cells other than eosinophils are also the effectors of adverse conditions in humans and animals as a result of stimulation by these cytokines and inhibition of such stimulation is also contemplated by this invention. These include cells that express either one or all of GM-CSF, IL-3 and IL-5 receptors, the stimulation of which leads to pathology. Examples of these are leukaemic cells, endothelial cells, breast cancer cells, prostate cancer cells, small cell lung carcinoma cells, colon cancer cells, macrophages in chronic inflammation such as rheumatoid arthritis, dendritic cells for immunosuppression and neutrophils in inflammation.
Thus in one form the invention may be said to reside in an inhibitor of leukaemic cell growth wherein the inhibitor is capable of inhibiting the binding of one or all of IL-3, GM-CSF and IL-5 to the xcex2c receptor. The inhibitor may be BION-1 or an agent capable of inhibiting BION-1 binding with xcex2c.
A number of different facets of eosinophil function might be modified so that in one form IL-5, IL-3 and GM-CSF mediated eosinophil survival is inhibited or blocked. In a second form IL-5, IL-3 and GM-CSF mediated eosinophil activation is inhibited or blocked.
In one form of this second aspect of the invention the monoclonal antibody or fragment thereof binds to at least the Fxe2x80x2-Gxe2x80x2 loop of domain 4 of the xcex2c subunit.
In an alternative form the monoclonal antibody or fragment thereof binds to at least the Bxe2x80x2-Cxe2x80x2 loop of domain 4 of the xcex2c subunit but this alternative form is not limited to monoclonal antibodies or fragments thereof that only bind to the Fxe2x80x2-Gxe2x80x2 loop but includes monoclonal antibodies or fragments thereof that perhaps binds to both the Fxe2x80x2-Gxe2x80x2 as well as the Bxe2x80x2-Cxe2x80x2 loop of domain 4 of the xcex2c.
It is thought that the monoclonal antibody isolated by the inventors inhibits dimerisation of the common receptor units and thus the invention might encompass an antibody or fragments thereof of the second aspect of the invention that inhibit xcex2c receptor dimerisation.
In one very specific form the monoclonal antibody is the antibody produced by the hybridoma cell line BION-1 (ATCC HB-12525).
In a broad form of a third aspect the invention could be said to reside in a hybridoma cell line capable of producing a monoclonal antibody of any form of the first or second aspect of the invention.
In one specific form of the third aspect of the invention the hybridoma cell line is BION-1 (ATCC HB-12525).
Since GM-CSF, IL-3 and IL-5 need to bind their respective xcex1 chains before being able to interact with xcex2c, at present most screening for new inhibitors utilise cell-based assays where both, xcex1 and xcex2c receptor units are co-expressed. Solid phase assays rely on inhibition of GM-CSF, IL-3 or IL-5 to their respective xcex1 chain only since these cytokines cannot bind to xcex2c alone. Since BION-1, unlike these three cytokines, can directly bind to xcex2c we propose that it can be used as a novel solid phase screening assay. Any compound that binds the appropriate site which is likely to inhibit all three cytokines will also inhibit the binding of BION-1. Additionally once further inhibitory compounds are uncovered these could be used in the place of BION-1 in that screening process. This therefore facilitates the screening of larger number of candidate inhibitor compounds.
In a broad form of a fourth aspect therefore the invention could be said to reside in a method of screening peptides, oligonucleotides and other small molecules for their capacity to competitively inhibit the binding of BION-1 or the binding of an agent capable of inhibiting BION-1 binding, to the xcex2c subunit.
Generally the screening assay involves contacting BION-1 or fragment thereof with the xcex2c subunit or fragment thereof as well as a candidate inhibitory compound, and measuring the degree of binding.
A reporting means is preferably provided to facilitate the detection of binding of BION1 or fragment thereof with xcex2c subunit or fragment thereof. Thus, for example, a competitive binding assay using labelled BION-1 could be used for this purpose, xcex2c or domain 4 of xcex2c is immobilized on a plate or tube and several compounds added, followed by labelled or tagged BION-1 or fragments thereof. Since BION-1 binds the region of xcex2c involved in binding all three cytokines, any compounds that block or reduce the binding of BION-1 or fragments thereof to xcex2c or domain 4 will be considered candidate inhibitory compounds. Thus, the availability of BION-1 as an agent that for the first time allows the direct binding to the cytokine binding region of xcex2c affords a novel test for the identification of simultaneous inhibitors of GM-CSF, IL-3 and IL-5. It will be understood that the same will apply for other cytokines and their respective receptors.
It will be understood that not the entire xcex2c subunit needs be used to screen candidate compounds, and certainly the present data indicates that a fragment of the xcex2c subunit encompassing domain 4 has sufficient structure in common with the native xcex2c subunit to reflect the configuration of the cellular target for an inhibitor useful for an in vivo effect.
In a broad form of a fifth aspect, the invention could be said to reside in a cytokine inhibitor capable of simultaneously blocking the binding of xcex2c by IL-3, GM-CSF, and IL-5 made according to the fourth aspect of the invention.
It is thought that compounds that inhibit binding of the IL-3, IL-5 and GM-CSF to the xcex2c will be therapeutically useful for intervention in conditions where IL-3, GM-CSF and IL-5 play a pathogenic role, mainly allergy, asthma, acute and chronic myeloid leukaemias, lymphoma and inflammation including rheumatoid arthritis, breast cancer and prostate cancer.
Similarly for other common cytokine receptors it is thought that antagonists or agonists will be therapeutically useful. gp130 is functionally analogous to xcex2c in that it is a common binding sub-unit and signal transducer for the IL-6, oncostatin M (OSM), ciliary neutrotrophic factor (CNTF), leukaemia inhibitory factor (LIF) and IL-11. It is suggested that raising an antibody against a domain analogous to domain 4 of xcex2c will also lead to blocking of two or more of these cytokines. Antagonism of this receptor system will be useful in inflammation, leukaemia and lymphoma. Antagonists to IL2Rxcex2/IL2Rxcex1 may be useful as immunosuppresants. Antagonists of LIFR may be useful for the prevention of implantation of embryos in uteri. Antagonists of IL-4/IL-13 will inhibit IgE production and may be useful in treating asthma and allergies.